P320: TCF3-PBX1 LEUKEMIA INVADES IPSC-DERIVED CEREBRAL ORGANOIDS
Authors: P. Gebing, S. Hänsch, L. Lenk, D. Schewe, A. Borkhardt, U. Fischer, S. Bhatia
Journal: HemaSphere (2022)

Background: Infiltration to the central nervous system (CNS) remains a major clinical challenge to this day in treating childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The relevant biological mechanisms underlying the invasion of the CNS, are not yet understood and are mainly investigated using 2D cell culture and in vivo mouse models1. Differences between species exist, particularly in cell types and architecture between human and murine CNS, as well as the lack of specificity of infiltration between leukemia subtypes. Human brain organoids potentially address some of these drawbacks and may therefore present a complemental approach to study CNS invasion. Aims: We aimed to establish human iPSC-derived cerebral organoids2 as a 3D model for CNS invasion. Applying these organoids in coculture with primary patient-derived xenograft (PDX) BCP-ALL cells (TCF3-PBX1), we aimed to investigate CNS-specific engraftment while using cord-blood-derived CD34-positive hematopoietic stem cells as a healthy control. Methods: Brain organoids were derived from induced pluripotent cell lines (iPSCs) of different donors and differentiated for 30 and 60 days. Intermediate progenitor, as well as matured neuronal cell types, were controlled by immunofluorescent staining of their respective markers (PAX6/Nestin, TUJ1/MAP2) which assemble in the complex neuroepithelium of cerebral organoids. We cocultured CFDA-SE stained TCF3-PBX1-positive patient-derived leukemia cells and cell lines with cerebral organoids. The initial co-cultivation was designed to investigate when/if infiltration could occur. Furthermore, a limiting dilution was introduced to determine the lowest cell concentration required for invasion. 3D imaging of cleared organoids was performed to analyze invasion. Results: Patient-derived TCF3-PBX-leukemic xenografted cells and cell lines invade cerebral organoids after 14 days of coculture. We found that this is independent of the iPSCs donors (testing in different batches an iPSC donors). The invasiveness was determined by quantifying single and larger colonies of cells engrafted into the organoids. TCF3-PBX1 revealed high invasiveness (P-value: 0.0006) compared to normal cord-blood-isolated CD34+ hematopoietic stem cells (HSCs). In addition, as few as 50 cells (TCF3-PBX1) per organoid were sufficient to invade the organoid tissue. Interestingly, the shRNA-downregulation of CD79a/Igα in TCF3-PBX1 (697) cells, impeded CNS infiltration to cerebral organoids (P-value: 0.0006), annealing their results in murine models1. Image:Summary/Conclusion: Human iPSC derived brain organoids display some key specificity with regards to of BCP-ALL CNS infiltration. Although key features (vasculature, leptomeninges etc.) which could strengthen the model, have not yet been successfully developed for brain organoids, they may present a promising 3D culture system to adopt in CNS infiltration studies. References: 1. Lenk, L. et al. Commun. Biol.4, 73 (2021). 2. Lancaster, M. A. et al. Nature501, 373–379 (2013).